DE69729125T2 - TROPONIN SUB-UNITS AND FRAGMENTS FOR USE AS INHIBITORS OF ANGIOGENESIS - Google Patents

Abstract

Pharmaceutical compositions and methods of treatment of diseases or disorders involving angiogenesis with therapeutically effective amounts of troponin C, I, or T, subunits, fragments, or analogs thereof.

Description

1 Introduction

The The present invention provides a novel pharmaceutical composition ready, and a method of using it for the treatment of diseases or disturbances, in which an abnormal angiogenesis is involved.

In In more detail, the present invention is based in part on the Discovery that the troponin subunits C, I and T proliferation of stimulated endothelial cells. It will be pharmaceutical Compositions containing therapeutically effective amounts of troponin subunits C, I or T, and methods of therapeutic use thereof provided.

2. Background

angiogenesis, the process of development and formation of new blood vessels, plays in numerous physiological processes, both normal and pathological, an important role. Angiogenesis takes place in response takes place on specific signals and involves a complex process by infiltration of the basal layer by vascular endothelial cells in response to one or more angiogenic growth signals, migration the endothelial cells in the direction of the source of the signal (s), and subsequent proliferation and formation of the capillary is. Blood flow through the newly formed capillary is initiated, after the endothelial cells with an already existing capillary have come into contact and have associated with it.

The Naturally present balance between endogenous stimulators and inhibitors angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., 1989, Cell 56: 345-355. In rare cases, at which a new formation of vessels under normal physiological conditions, such as wound healing, Regeneration of organs, embryonic development and reproductive processes of the Woman, angiogenesis is strictly regulated and temporally and spatially limited. Under conditions of pathological angiogenesis, such as those which mark the growth of a solid tumor, these fail regulatory controls.

A unregulated angiogenesis becomes pathological and supports one Progression of many neoplastic and non-neoplastic diseases. A variety of serious diseases, including growth solid tumors and metastases, arthritis, some types of eye diseases and psoriasis, are dominated by abnormal neoplasm of vessels. See, for example, B. Review article by Moses et al., 1991, Biotech 9: 630-634; Folkman et al., 1995, N. Engl. J. Med. 333: 1757-1763; Auerbach et al., 1985, J. Microvasc. Res. 29: 401-411; Folkman, 1985, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pages 175-203; Patz, 1982, Am. J. Ophthalmol. 94: 715-743; and Folkman et al., 1983, Science 221: 719-725. The process of angiogenesis contributes to a number of pathological conditions to the disease state. For example, significant data has been accumulated which suggest that the growth of solid tumors from angiogenesis depends. Folkman and Klagsbrun, 1987, Science 235: 442-447.

The Preservation of the vascularity The cornea, the lens, and the trabecular network are responsible for both vision and vision also for the physiology of the eye is essential. There are several eye diseases, many of which lead to blindness, in which, in response to the disease state, a neovascularization in the Eye takes place. These eye diseases include diabetic retinopathy, neovascular Glaucoma, inflammation and ocular tumors (e.g., retinoblastoma). There are also a number from other eye diseases, which also coincide with the neoplasm of Associated vessels include, retrolental fibroplasia, uveitis, prematurity retinopathy, macular Degeneration, and approximately twenty eye diseases associated with choroidal vascular disease are associated and approximate forty eye diseases associated with vascularization of the iris are. See, for example, B. Review article by Waltman et al., 1978, Am. J. Ophthal. 85: 704-710 and Gartner et al., 1978, Surv. Ophthalmolo. 22: 291-312. Currently the treatment of these diseases, especially after a neovascularization has taken place, inadequate and blindness often results. Investigations have suggested that vasomotor-inhibiting factors present in normal ocular tissue (cornea and vitreous body) are present, have been lost in the diseased state.

An inhibitor of angiogenesis could play a significant therapeutic role in contributing to the pathological progression of the underlying disease states and provides a valuable means of investigating their causes of illness. For example, agents that inhibit neovascularization of tumors could play an important role in inhibiting the growth of metastatic tumors.

It It was found that the components of angiogenesis, the with the proliferation, migration and invasion of vascular endothelial cells in part regulated by polypeptide growth factors become. Experiments in culture indicate that endothelial cells, exposed to a medium containing suitable growth factors can be induced to cause some or all of the angiogenic reactions. There are identified several polypeptides having in vitro endothelial growth promoting activity Service. Examples include the acidic and basic fibroblast growth factor, the transforming growth factors α and β, the platelet-derived endothelial cell growth factor, the granulocyte colony stimulating factor, Interleukin-8, the Hepatocyte growth factor, Proliferin, the vascular Endothelial growth factor and the placental growth factor. See, for example, B. the review article by Folkman et al., 1995, N. Engl. J. Med. 333: 1757-1763.

Even though It has been shown that extracts of several different Tissue sources containing anti-angiogenic activity, found out was that several molecules, such as platelet factor-4, thrombospin, Protamine and the transforming growth factor B different Aspects of angiogenesis, such as cell proliferation or cell migration, Negatively regulate is not a single one in the known art tissue-derived macromolecule has been identified which is capable to inhibit angiogenesis. See, for example, B. Review article by Folkman J., 1995, N. Engl. J. Med. 333: 1757-1763, and D'Amore, 1985, Prog. Clin. Biol. Res. 221: 269-283. There is thus a big one Need for the further identification and characterization of chemical Means, which is the continued deregulated spread of neovascularization can prevent and the potentially broad applicability as a therapy for those Diseases in which a neoplasm of vessels is a predominant role plays, could have.

capillary Endothelial cells ("EC") proliferate during the New formation of vessels in response to an angiogenic stimulus. Ausprunck and Folkman, 1977, J. Microvasc. Res. 14: 153-65. An in vitro assay showing endothelial cell proliferation in response to known angiogenesis stimulating factors, such as the acidic or basic fibroblast growth factor (aFGF or bFGF), evaluated, was developed around the process of neoplasm of vessels in vitro imitate. This type of assay is the assay of choice to the Stimulation of proliferation of capillary EC by various to show angiogenic factors. Shing et al., 1984, Science 223: 1296-1298.

Of the Process of capillary EC migration through the extracellular matrix to angiogenic stimulus is also critical for angiogenesis required event. See, for example, B. the review article of Ausprunck et al., 1977, J. Microvasc. Res. 14: 53-65. This process presents An additional Assay to mimic the process of neovascularization in vitro ready. A modification of the Boyden chamber technique has been developed to monitor the EC migration. Boyden et al., 1962, J. Expt. Med. 115: 453-456, Example 4. So far Only a few tissue-derived inhibitors of EC cell migration are known. See, for example, B. the review article Langer et al., 1976, Science 193: 70-72.

In the early one Seventies has been a number of in vivo angiogenesis model bioassays widely used. These model systems included the bioassays Rabbit cornea pocket, chicken chorionic allantoic membrane (chicken chorioallantoic membrane, "CAM"), Rat dorsal air sac (rat dorsal air sac) and rabbit air chamber (rabbit air chamber). For an overview, see Blood et al., 1990, Biochem, et Biophys. Acta 1032: 89-118. The Development of controlled release polymers which are capable of size molecules how to release stimulators and inhibitors of angiogenesis, was for the use of these assays critically. Langer et al., 1976, Nature 263: 797-800.

The CAM bioassay cultured fertilized chicken embryos in petri dishes. On day 6 of development, a disc of a release polymer, such as methylcellulose, impregnated with the test sample or a suitable control substance is placed on the vessel membrane, on its leading edge. On day 8 of development, the area around the implant is observed and evaluated. Vascular free zones surrounding the test implant indicate the presence of an inhibitor of embryonic neovascularization. Moses et al., 1990, Science 248: 1408-1410, and Taylor et al., 1982, Nature 297: 307-312. The published dosages for previously described inhibitors of angiogenesis, tested alone in the CAM assay, are 50 μg protamine (Taylor et al. (1982)), 200 μg bovine vitreous humor extract (Lutty et al., 1983, Invest. Ophthalmol. Vis. Sci., 24: 53-56), and 10 μg platelet factor IV (Taylor et al., 1982). The lowest published dosages of Inhibitors of angiogenesis, acting as combinations, include heparin (50 μg) and hydrocortisone (60 μg), and B-cyclodextrin tetradecasulfate (14 μg) and hydrocortisone (60 μg), published by Folkman et al., 1989, Science 243: 1490.

According to the rabbit corneal bag assay are polymer pellets of ethylene vinyl acetate copolymer ("EVAC") with test substance waterproof and surgically in a pocket in the rabbit cornea about 1 mm implanted from the limbus. Langer et al., 1976, Science 193: 707-72. Around testing for an inhibitor of angiogenesis will either be one Piece of carcinoma or another angiogenic stimulant distal to the polymer 2 mm implanted from the limbus. Become in the other eye of each rabbit Control polymer pellets, which are empty, in the same way in nearby implanted with an angiogenic stimulant. In these control grains start after 5-6 Days capillary blood vessels in the direction of the tumor implant, eventually growing over the empty polymer. The targeted growth of new capillaries occurs in the test kernels out of the limbus blood vessel in the direction of the tumor at a reduced rate and is often inhibited, so that a vascular free Area is observed around the polymer around. This assay will quantified by measuring the maximum vessel lengths with a stereospecific Microscope.

Troponin, a complex of three polypeptides, is an accessory protein that is closely associated with actin filaments in vertebrate muscle. The troponin complex acts in conjunction with the muscle form of tropomyosin to mediate the Ca ²⁺ dependence of myosin ATPase activity and thereby regulate muscle contraction. The troponin polypeptides T, I and C are named for their tropomyosin binding, inhibition, and calcium binding activities, respectively. Troponin T binds to tropomyosin and is thought to be responsible for the positioning of the troponin complex on the thin muscle fiber. Troponin I binds to actin, and the complex formed by troponin I and T and tropomyosin inhibits the interaction of actin and myosin. Troponin C can bind up to four calcium molecules. Studies suggest that when the calcium concentration in the muscle is increased, troponin C causes troponin I to lose its hold on the actin molecule, which causes tropomyosin molecular rearrangement, exposing the myosin binding sites to actin and stimulating myosin ATPase activity becomes. Prior to the discovery of the present invention, it has not been known that troponin subunits inhibit the process of endothelial cell proliferation.

WHERE 91/1193 describes pharmaceutical compositions for inhibition angiogenesis comprising a collagenase inhibitor, in particular one of cartilage derived inhibitor with a defined N-terminal amino acid sequence. This was based on the discovery that inhibition of collagenase alone can inhibit angiogenesis.

The Citing a document herein is not to be construed as a concession be that such a document for the present invention known prior art.

3. Summary of the Fiction

The The present invention relates to pharmaceutical compositions, which are the troponin subunits C, I or T or fragments thereof contained in therapeutically effective amounts, the proliferation of endothelial cells. The invention also relates to pharmaceutical compositions which Analogs of troponin subunits C, I or T in therapeutic containing effective amounts, the proliferation of endothelial cells can inhibit. The invention further relates to the treatment of disorders in Associated with the rebuilding of vessels by administering a therapeutic compound of the invention. Such therapeutic Compounds (referred to herein as "therapeutics") include: the troponin subunits C, I and T and fragments and analogs from that. In one embodiment a therapeutic agent according to the invention administered to treat a cancerous condition, or to the progression from the pre-neoplastic or premalignant Condition to prevent a neoplastic or malignant condition. In other specific embodiments becomes a therapeutic agent according to the invention administered to eye diseases associated with neoplasm of vessels are to be treated.

3.1 Definitions

As used herein:

Of the Term "troponin subunit", unless the Designations C, I or T preceded generically means each the troponin subunits C, I or T.

4. Short description the figures

1 , Inhibition of proliferation of bovine capillary endothelial cells (BCE) by troponin C. The percent inhibition of bFGF-stimulated BCE proliferation is shown as a function of troponin C concentration (nM). Percent inhibition was determined by comparing results obtained for stimulus-only cells with those obtained for samples exposed to both stimulus and inhibitor. The test volume was 200 μl.

2 , Inhibition of Capillary BCE Proliferation by Troponin I. Percent inhibition of bFGF-stimulated BCE proliferation is shown as a function of troponin I concentration (nM). The percent inhibition was determined as to 1 described. The test volume was 200 μl.

3 , Inhibition of capillary BCE proliferation by troponin T. Percent inhibition of bFGF-stimulated BCE proliferation is shown as a function of troponin T concentration (nM). The percent inhibition was determined as to 1 described. The test volume was 200 μl.

4 , Inhibition of BCE proliferation by troponin C and I. The percent inhibition of bFGF-stimulated BCE proliferation is shown as a function of the concentration of troponin I and C (nM). The percent inhibition was determined as to 1 described. The test volume was 200 μl.

5 , Inhibition of capillary BCE proliferation by troponin C, I and T. The percent inhibition of bFGF-stimulated BCE proliferation is shown as a function of the concentration of troponin C, I and T (nM). The percent inhibition was determined as to 1 described. The test volume was 200 μl.

6 , Inhibition of tumor growth in SCID mice.

5. Detailed Description of the invention

The present invention relates to therapeutic methods and compositions based on troponin subunits. The invention provides treatment of disorders associated with the rebuilding of vessels by administering a therapeutic compound of the invention. Such therapeutic compounds (referred to herein as "therapeutics") include: the troponin subunits C, I and T, fragments and analogs thereof (collectively "peptides of the invention"). The peptides of the invention are characterized by the property of inhibiting the proliferation of bovine endothelial cells in culture with an IC ₅₀ of 10 μM or smaller. In a preferred embodiment, a therapeutic agent of the present invention is administered to treat a cancerous condition or to prevent the progression from the preneoplastic or non-malignant condition to a neoplastic or malignant condition. In other specific embodiments, a therapeutic agent of the present invention is administered to treat an eye disease associated with neoplasm of vessels.

In In a preferred aspect, a therapeutic agent of the invention is a peptide, which consists of at least one fragment of troponin C, troponin I, Troponin T or troponins C and I, and which is effective is to inhibit the proliferation of endothelial cells.

Examples the troponin subunits used according to the invention can, include the troponin subunits from human fast-twitch skeletal muscle, the sequences of which are given below are:

Human Fast Twitch Skeletal Muscle Troponin C (SEQ ID NO: 1)

Human Fast Twitch Skeletal Muscle Troponin I (SEQ ID NO: 2)

Human Fast Skeleton Beta Troponin T (SEQ ID NO: 3)

In another embodiment For example, the invention encompasses peptides resulting in human fast-twitch skeletal troponin C (SEQ ID NO: 1) are homologous. In one embodiment, the amino acid sequence has at least 80% identity of the peptide compared to the fragment of human fast-twitch skeletal troponin C, from which it derives is (the "prototype fragment"). In another embodiment is this identity more than 85%. In a stronger preferred embodiment is this identity more than 90%. In a most preferred embodiment has the amino acid sequence of the peptide at least 95% identity with the prototype fragment. Fragments can have at least 75, 100 or 120 amino acids.

In another embodiment For example, the invention encompasses peptides resulting in human fast-twitch skeletal troponin I (SEQ ID NO: 2) are homologous. In one embodiment, the amino acid sequence has At least 80% of the peptide's identity with the human fast-twitch Skeletal troponone I prototype fragment. In another embodiment is this identity more than 85%. In a stronger preferred embodiment is this identity more than 90%. In a most preferred embodiment has the amino acid sequence of the peptide at least 95% identity with the prototype fragment. Fragments can have at least 75, 100 or 120 amino acids.

In another embodiment For example, the invention encompasses peptides resulting in human fast-twitch skeletal troponin T (SEQ ID NO: 3) are homologous. In one embodiment, the amino acid sequence has At least 80% of the peptide's identity with the human fast-twitch Skeleton beta troponin T prototype. In another embodiment is this identity more than 85%. In a stronger preferred embodiment is this identity more than 90%. In a most preferred embodiment has the amino acid sequence of the peptide at least 95% identity with the prototype fragment. Fragments can a length of at least 75, 100, 120 or 200 amino acids.

In other specific embodiments are the peptides of the invention Troponin C, troponin I and troponon T subunits of the fast-twitch, slow-twitch and cardio isoforms of other mammalian species, e.g. Human, Rabbit, rat, mouse, cattle, sheep and pig.

In a specific embodiment becomes a therapeutic agent according to the invention combined with a therapeutically effective amount of another molecule, which negatively regulates angiogenesis, which may be not limited is on platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16 kD fragment), angiostatin (38 kD fragment of plasminogen), bfGf more soluble Receptor, transforming growth factor β, interferon alpha, and placental-proliferin-related Protein.

Paradoxically the neoplasm of vessels of a tumor gradually diminishes the Accessibility of chemotherapeutic drugs, due to increased interstitial pressure within the tumor, causing vascular compression and central necrosis causes. In vivo results have shown that rodents receiving angiogenic therapy have an increased delivery chemotherapy to a tumor. Teicher et al., 1994, Int. J. Cancer 57: 920-925. Thus, in one embodiment, the invention provides a pharmaceutical Composition of the present invention in combination with a chemotherapeutic agent ready.

In Another preferred aspect is a therapeutic agent according to the invention with chemotherapeutic agents or treatment with one radioactive isotope combined.

The Invention is illustrated below by way of examples, which, inter alia, the inhibition of the proliferation of capillary Endothelial cells through the troponin subunits C, I and T, and the means of determining the inhibition of migration of capillary Endothelial cells and the inhibition of neovascularization in vivo revealed by troponin subunits.

Out establish clarity of the disclosure and is in no way limiting the detailed Description of the invention in the subsections set out below divided.

5.1 TROPONINE SUB-UNITS, FRAGMENTS AND ANALOG

The The invention provides pharmaceutical compositions which Troponin subunits include. In special aspects are the Subunits troponin subunits from fly, frog, mouse, rat, rabbit, pig, cattle, dog, Monkey or human.

you can imagine that troponin subunits are produced can by modifying Troponin sequences by substitutions, additions or deletions that are functionally equivalent molecules provide. These include, but are not limited to Troponin subunits, which as a primary amino acid sequence contain all or part of the amino acid sequence of a troponin subunit, comprehensively changed Sequences in which residues within the sequence are functionally equivalent amino acid residues exchanged, resulting in a silent exchange. For example can one or more amino acid residues within the sequence by another amino acid with a similar polarity, called a functional equivalent works, be replaced, which leads to a silent exchange. replacement substances for one amino acid within the sequence from the other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, Leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and Glutamine. The positively charged (basic) amino acids include Arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

A embodiment The invention provides molecules ready, consisting of a fragment of at least 10 (consecutive) amino acids a troponin subunit consist of or include the proliferation of endothelial cells can inhibit. In other embodiments is this molecule from at least 20 or 50 amino acids the troponin subunit. In specific embodiments exist such molecules or include such molecules Fragments of a troponin subunit of at least 75, 120 or 200 amino acids.

In a preferred embodiment the protein is a mammalian troponin subunit. In alternative embodiments it is a mammalian troponin subunit C, I or T.

The fragments and analogs of troponin subunits of the invention may be derived from tissue (see, for example, Example 1, Ebashi et al., 1968, J. Biochem 64: 465; Yasui et al., 1968, J. Biol. Chem. 243: 735 Hartshorne et al., 1968, Biochem Biophys Res. Comm 31: 647; Shaub et al., 1969, Biochem J. 115: 993; Greaser et al., 1971, J. Biol. Chem. 246: 4226 Chem. 251: 866-871; and Yates et al., 1983, J. Biol. Chem. 258: 5770-5774), or by various means, in the art produced known methods. The manipulations that lead to their production can be at the level of the gene or the protein. For example, a cloned troponin gene sequence encoding the troponin subunits C, I or T can be modified by any of numerous strategies known in the art. Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. The sequence may be appropriate Sites with one or more restriction endonucleases, followed by further enzymatic modification, if desired, isolated and ligated in vitro. In preparing a gene encoding a derivative or analog of a troponin subunit, care should be taken to ensure that the modified gene within the gene region encoding the desired troponin activity is in the same translation reading frame as the troponin gene Subunit remains and is not interrupted by translation stop signals.

In addition, the troponin subunit-encoding nucleic acid sequence can be mutated in vitro or in vivo to create and / or destroy translation, initiation and / or termination sequences, or to create variations in coding regions and / or to introduce new restriction endonucleases or to destroy existing ones to facilitate further in vitro modification. Any known in the art mutagenesis technique can be used, including, but not limited to in vitro site-directed mutagenesis (Hutchinson et al, 1978, J. Biol Chem. 253:.. 6551), use of TAB ^® linkers (Pharmacia), etc.

Manipulations of the sequence of the troponin subunit C, I or T can also be performed at the level of the protein. Included within the scope of the invention are troponin subunits which have been differentially modified during or after translation, e.g. By acetylation, phosphorylation, carboxylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be made by known techniques, including but not limited to to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ₄ , acetylation, formylation, oxidation, reduction, etc.

In addition, troponin subunits be chemically synthesized. For example, a peptide that corresponds to a part of a troponin subunit comprising the desired domain, or which in vitro the desired activity mediated, synthesized using a peptide synthesizer. Furthermore, if desired, non-classical amino acids or chemical amino acid analogs as a substitution or addition into the sequence of a troponin subunit introduced become. Non-classical amino acids include, but are not limited to to the D-isomers of the usual Amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, hydroxyproline, Sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, Phenylglycine, cyclohexylalanine, β-alanine, designer amino acids such as β-methyl amino acids, Ca methyl amino acids and Na-methyl amino acids.

In a specific embodiment the invention comprises a chimeric one Protein, or fusion protein, comprising a troponin subunit, at their amino or carboxy terminus via a Peptide bond to an amino acid sequence a different protein is bound. Such a thing chimeric product can be made by the appropriate, for the desired Coding amino acid sequences nucleic acid sequences in the correct reading frame are ligated together, according to in the Technique known methods, and the chimeric product by methods, which are well known in the art. alternative can be such a chimeric Product produced by protein synthesis techniques, for. B. through the use of a peptide synthesizer.

5.2 ASSAYS OF TROPONIN PROTEINS, FRAGMENTS AND ANALOG

The functional activity and / or therapeutically effective dose of troponin subunits, Fragments and analogues can be synthesized by different methods in vitro be tested. These methods are based on angiogenesis involved physiological processes, and although they are to the extent of protection belonging to the invention, they should not restrict the methods by which troponin subunits, fragments and analogs that inhibit angiogenesis and / or a therapeutically effective dosage of the pharmaceutical Composition is determined.

If for example, on the ability of troponin subunits, Fragments and analogues being tested, the proliferation of capillary To inhibit or impair endothelial cells (EC) in vitro, can various bioassays known in the art are used, including but not limited to radioactive incorporation into nucleic acids, colorimetric assays, and counting of cells.

Inhibition of endothelial cell proliferation can be measured by colorimetric determination of cellular acid phosphatase activity or electronic cell counting. These methods provide a rapid and sensitive screening method to estimate the number of endothelial cells in culture after treatment with the troponin subunit, the derivative or analog of the invention and an angiogenesis-stimulating factor such as aFGF. The colorimetric determination of the activity of cellular acid phosphatase is described by Connolly et al., 1986, J. Anal. Biochem. 152: 136-140. According to this method, described in Example 3, capillary endothelial cells are treated with angiogenesis-stimulating factors such as aFGF and a range of potential inhibitor concentrations. These samples are incubated to allow growth and then harvested, washed, lysed in a buffer containing a phosphatase substrate, and then incubated a second time. A basic solution is added to stop the reaction and color development is determined at 405λ. According to Connolly et al. For up to 10,000 cells / sample, a linear relationship between the activity of acid phosphatase and the number of endothelial cells is obtained. Standard curves for the activity of acid phosphatase in known numbers of cells are also generated to confirm that the enzyme values represent the actual numbers of EC. Percent inhibition is determined by comparing the number of cells from samples exposed to the stimulus with those exposed to both stimulus and inhibitor.

colorimetric Assays for determining the effect of troponin subunits C, I and T on the proliferation of endothelial cells show that all three troponin subunits affect the proliferation of bFGF-stimulated endothelial cells.

Troponin C inhibited the proliferation of bFGF-stimulated endothelial cells in a dose-dependent manner at all concentrations tested ( 1 ). The percent inhibition of bovine endothelial cell proliferation ("BCE") was 54%, 86%, 83% and 100%, respectively, at concentrations of 280 nM, 1.4 μM, 2.8 μM and 5.6 μM, respectively. An inhibition of 100% was observed at a concentration of 20 μg / well (5.6 μM). IC ₅₀ represents the concentration at which a 50% inhibition of growth factor aFGF-induced stimulation was observed. It was determined that the IC ₅₀ of troponin C is 278 nM.

Troponin I inhibited bFGF-stimulated BCE proliferation in concentrations of 1 and 5 μg / well, but no inhibition was observed in the sample tested at 10 μg / well ( 2 ). The percent inhibition of BCE was 33% and 46%, respectively, at concentrations of 240 nM and 1.2 μM, respectively. It was determined that the IC ₅₀ of troponin I is 1.14 μM.

Troponin T inhibited bFGF-stimulated EC proliferation at concentrations of 10 and 20 μg / well, but not at concentrations of 1 and 5 μg / well ( 3 ). The proliferation of BCE was inhibited at 1.6 μM and 3.3 μM, respectively, at 23% and 62%, respectively. The IC ₅₀ of troponin T was determined to be 2.14 μM.

The combination of troponin subunits C and I inhibited EC at all concentrations tested ( 4 ). The percent inhibition of BCE was 52%, 54%, 73% and 47% at 130 nM, 645 nM, 1.3 μM and 2.6 μM, respectively. It was determined that the IC _{50 of} this combination is 110 nM.

It has been observed that the combination of troponin subunits C, I and T at a concentration of 360 nM (5 μg / well, 5 ) inhibited aFGF-stimulated BCE proliferation by 16%.

The tested troponin samples had no detectable inhibitory Effect on the growth of Balb / c 3T3 cells, a non-endothelial Cell type.

The incorporation of radioactive thymidine by capillary endothelial cells provides another means of testing for the inhibition of endothelial cell proliferation by a potential angiogenesis inhibitor. According to this method, a predetermined number of capillary endothelial cells are cultured in the presence of a ³ H thymidine stock, an angiogenesis stimulator such as, for example, bFGF, and a range of concentrations of the angiogenesis inhibitor to be tested. After incubation, the cells are harvested and the extent of thymidine incorporation determined. See example 2.

The ability of varying concentrations of troponin subunits, fragments or analogues, the process of migration of capillary endothelial cells in response to an angiogenic stimulus, can be tested using the modified Boyden chamber technique become. See Section 2 and Example 4 below.

Another means to test the functional activity of troponin subunits, fragments, and analogs involves testing the compounds for the ability to target directed migration of capillaries Endothelial cells, which ultimately leads to the formation of capillary tubes to inhibit. This ability can be evaluated, for example, using an assay in which capillary endothelial cells plated on collagen gels are stimulated with the inhibitor and it is determined whether capillary-like tubular structures are formed by the cultured endothelial cells.

assays on the ability To inhibit angiogenesis in vivo include the chicken chorionic allantoic membrane assay see section 2, and Example 5, below) and the rat or rabbit corneal bag assays. See Polverini et al., 1991, Methods Enzymol. 198: 440-450. According to the corneal bag assays becomes a tumor of choice in the cornea of the test animal in the mold implanted in a corneal pocket. The potential angiogenesis inhibitor is applied to the corneal pouch and the corneal pouch becomes routinely investigated the neoplasm of vessels. See Section 2, and Example 6, below.

A embodiment invention provides a combination of troponin subunits, Fragments or analogs of the present invention ready for angiogenesis to inhibit. Another embodiment provides the combination of troponin subunits, fragments or analogs with other factors inhibiting angiogenesis. such angiogenesis inhibitory factors include, but are not limited on: angiostatin steroids, Thrombospondin, platelet factor IV, transforming growth factor β, interferons, tumor necrosis factor α, bovine vitreous extract, Protamine, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), Prolactin (16 kD fragment), angiostatin (38 kD fragment of plasminogen), the bfGf soluble Receptor, and placental-proliferin-related Protein. See, for example, B. Review article of Folkman et al., 1995, N. Engl. J. Med. 333: 1757-1763, and Klagsbrun et al., 1991, Annu. Rev. Physiol. 53: 217-239.

The therapeutically effective dosage for the inhibition of angiogenesis in vivo, defined as the inhibition of proliferation, migration of capillary endothelial cells and the ingrowth of blood vessels from in vitro inhibition assays using the above Compositions of the invention or in combination with other angiogenesis inhibiting factors be extrapolated. The effective dosage will also depend on the method of administration and the administering agent. For example, in some Applications, such as in the treatment of psoriasis or diabetic Retinopathy, the inhibitor administered in a topical ophthalmic carrier. In other applications, such as in the treatment of solid tumors, the inhibitor is produced by means of a biodegradable polymer Implant administered. The protein can also be modified for example, by polyethylene glycol treatment.

5.3 THERAPEUTIC USES

The Invention provides a treatment of diseases or disorders, which are associated with a new formation of vessels, ready, by administering a therapeutic compound of the invention. such Therapeutic compounds (referred to herein as "therapeutics") include troponin subunits and fragments and analogs thereof (e.g., as described below).

5.3.1 MALIDICAL DISEASES

Malignant and metastatic states, which with the therapeutic compounds of the present invention can be treated include, but are not limited to to the solid tumors listed in Table 1 (for a review of such diseases, see Fishman et al., 1985, Medicine, 2. Edition, J.B. Lippincott Co., Philadelphia).

TABLE 1

MALIGN AND RELATED DISEASES

Solid tumors

Sarcomas and carcinomas

• fibrosarcoma
• myxosarcoma
• liposarcoma
• chondrosarcoma
• Osteogenic sarcoma
• chordoma
• angiosarcoma
• endotheliosarcoma
• lymphangiosarcoma
• Lymphangioendothelsarkom
• synovioma
• mesothelioma
• Ewing's tumor
• leiomyosarcoma
• rhabdomyosarcoma
• colon cancer
• pancreatic cancer
• breast cancer
• Ovarian Cancer
• Prostate Cancer
• Squamous cell carcinoma
• basal cell carcinoma
• adenocarcinoma
• sweat glands carcinoma
• sebaceous gland carcinoma
• papillary
• Papillenadenokarzinome
• cystadenocarcinoma
• Medullokarzinom
• Bronchogenic carcinoma
• Renal cell carcinoma
• hepatoma
• Cholangiocarcinoma
• choriocarcinoma
• seminoma
• embryonal carcinoma
• Wilms tumor
• cervical cancer
• testicular tumor
• lung cancer
• Small cell lung carcinoma
• bladder cancer
• epithelial
• glioma
• astrocytoma
• medulloblastoma
• craniopharyngioma
• ependymoma
• Kaposi's sarcoma
• pinealoma
• hemangioblastoma
• Gehörneurom
• oligodendroglioma
• Menangiom
• melanoma
• neuroblastoma
• retinoblastoma

5.3.2 EYE DISEASES

Ocular diseases associated with neoplasm of vessels which may be treated with the therapeutic compounds of the present invention include, but are not limited to:
Neovascular glaucoma
Diabetic retinopathy
retinoblastoma
retrolental fibroplasia
uveitis
Retinopathy of prematurity
macular degeneration
Korneatransplantat neovascularization
as well as other inflammatory ocular diseases, ocular tumors and diseases associated with choroidal neoplasm of vessels or reformation of vessels of the iris. See, for example, B. reviewed by Waltman et al., 1978, Am. J. Ophthal. 85: 704-710, and Gartner et al., 1978, Surv. Ophthalmolo. 22: 291-312.

5.3.3 OTHER FAULTS

Other disorders which with the therapeutic compounds of the present invention can be treated include, but are not limited to on hemangioma, Arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, Granulations, hemophilic Joints, hypertrophic scars, poor fracture healing, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

5.4 PROOF OF A THERAPEUTIC OR PROPHYLACTIC FITNESS

The Therapeutics of the invention can in vivo to the desired therapeutic or prophylactic activity and for the determination of therapeutically effective dosage can be tested. For example can such compounds before testing in humans in suitable Animal model systems are tested, including but not limited to Rats, mice, Chicken, Cattle, monkeys, rabbits, etc. For In vivo testing before administration to humans can be done in any art known animal model system can be used.

5.5 THERAPEUTIC / PROPHYLACTIC ADMINISTRATION AND COMPOSITIONS

The Invention provides methods of treatment (and prophylaxis) by administering an animal an effective amount of an animal Therapeutic invention. According to one preferred aspect, the therapeutic is substantially purified, as stated in Example 1. The animal is preferably an animal, including but not limited to Animals such as cattle, pigs, chickens, etc., and is preferred a mammal and most preferably a human.

The The invention further provides methods of treatment by administering a living being of an effective amount of a therapeutic agent according to the invention in combination with a chemotherapeutic agent and / or treatment with a radioactive isotope ready.

The The invention also provides methods of treatment with a therapeutic agent of the invention for patients ready, where a remission has taken place to a dormant Condition to maintain.

It Different delivery systems are known and these can be used for Administration of a therapeutic agent according to the invention used be, for. B. encapsulation in liposomes, microparticles, microcapsules, Receptor-mediated endocytosis (see, eg, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432). Methods of introduction include, but are not limited to topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic and oral routes of administration. The connections can by every favorable Way, for example by infusion or bolus injection, by absorption through epithelial or mucosal linings (e.g. Oral mucosa, rectal mucosa and intestinal mucosa, etc.), and you can administered together with other biological agents. It is preferred that the administration be local, but they do can be systemic. additionally it may be desired be, the pharmaceutical of the invention To introduce compounds into the central nervous system any suitable route, including intraventricular and intrathecal injection; intraventricular Injection may be by an intraventricular catheter, for example connected to a reservoir, such as an Ommaya reservoir, be simplified. Administration to the lungs may also be be applied, for. B. by the use of an inhaler or atomizer, and formulation with an aerosol forming agent.

In a specific embodiment, it may be desirable to use the pharmaceutical composition of the invention locally to the area which requires treatment, to administer; this may be achieved, for example and not in a limiting sense, by local infusion during a surgical procedure, topical application, e.g. In conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, wherein the implant consists of a porous, non-porous or gel-like material comprising membranes such as sialastic membranes or fibers. In one embodiment, administration may be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.

For a topical Application, the purified troponin subunit is combined with a carrier, so that an effective dosage is delivered, based on the desired activity (i.e., in the range of an effective dosage of, for example, 1.0 μM to 1.0 mM to a localized angiogenesis, migration of endothelial cells to prevent and / or for an inhibition of the proliferation of capillary endothelial cells). In one embodiment becomes a troponin subunit, fragment or analog for treatment of diseases such as psoriasis applied topically to the skin. The carrier For example, and not in a limiting sense, in the form of Ointment, cream, gel, paste, foam, aerosol, suppository, Pillow or gel stick be.

One topical therapeutic for the treatment of some of those discussed below Eye diseases consists of an effective amount of a troponin subunit, a fragment or analog in an ophthalmologically acceptable Arzneimittelrräger such as buffered saline, Mineral oil, vegetable oils such as corn or arachis, Petroleum jelly, Miglyol 182, alcohol solutions, or liposomes or Liposome-like Products. Each of these compositions may also contain preservatives, Antioxidants, antibiotics, immunosuppressants, and other biological or pharmaceutically active agents which have no harmful effect on the troponin subunit.

For directional internal topical applications, such as the treatment of ulcers or hemorrhoids, may be the composition of the troponin subunit, the fragment or analogues in the form of tablets or capsules, which each of the following components or compounds of a similar Nature may contain: a binder such as microcrystalline cellulose, gum tragacanth or Gelatin; a drug carrier like strength or lactose; a disintegrant such as alginic acid, primogel or cornstarch; one Lubricants such as magnesium stearate or Sterotes, or a lubricant such as colloidal silica. When the dosage form is a Capsule is, it can be added to material of the above type containing a liquid carrier such as a fatty oil. additionally can Unit dosage forms contain various other materials, which modify the physical form of the unit dosage, for example Coatings of sugar, shellac or other enteric agents.

Suppositories (Suppositories) generally contain active ingredient in the range of 0.5% by weight to 10% by weight; oral formulations preferably contain 10% to 95% Active ingredient.

In another embodiment The therapeutic may be in a vesicle, especially a liposome be delivered. See Langer et al., 1990, Science 249: 1527-1533; treat et al., 1989, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (Eds.), Liss, New York, Pages 353-365; Lopez Berestein, a. a. O., pages 317-327.

In a still different embodiment The therapeutic agent can be used in a controlled-release system be delivered. In one embodiment, for administration the troponin subunit uses an infusion pump, such as, for example, those used to deliver insulin or chemotherapy to specific organs or tumors (See Langer, supra; Sefton, CRC Crit Ref. Biomed. Closely. 14: 201; Buchwald et al., 1980, Surgery 88: 507; Saudek et al. 1989, N. Engl. J. Med. 321: 574).

In a preferred form, the troponin subunit is administered in combination with a biodegradable biocompatible polymeric implant which releases the troponin subunit at a selected site for a controlled period of time. Examples of preferred polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate and copolymers and mixtures thereof. See Medical Applications of Controlled Release, Langer and Wise (eds), 1974, CRC Pres., Boca Raton, Florida; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (ed.), 1984, Wiley, New York; Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 71: 105. In yet another embodiment, a system with controlled release in the vicinity of the therapeutic target, ie, the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, 1989, supra, Vol 115-138).

Other Controlled release systems are reviewed in Langer's review (1990, Science 249: 1527-1533) discussed.

The The present invention also provides pharmaceutical compositions ready. Such compositions comprise a therapeutic effective amount of a therapeutic, and a pharmaceutically acceptable Carrier.

The Pharmaceutical compositions of the invention may be described as neutral forms or salt forms are formulated. pharmaceutical Acceptable salts include those containing free amino groups are formed, such as those which of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc. and those having free carboxyl groups such as those derived from sodium hydroxide, potassium hydroxide, Ammonium hydroxide, calcium hydroxide, iron hydroxide, isopropylamine, Triethylamine, 2-ethylaminoethanol, Histidine, procaine, etc. are derived.

In a specific embodiment the term "pharmaceutically acceptable "recognized by an approval authority the federal government or the government of a state, or listed in the US Pharmacopoeia or another generally accepted pharmacopoeia for use in animals and especially in humans. The term "carrier" denotes a diluent, Adjuvant, a drug carrier or a vehicle with which the therapeutic is administered. such pharmaceutical carriers can sterile liquids such as water and oils, including those of petroleum, animals, plants or synthetic Origin, such as peanut oil, Soybean oil, Mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier, when the pharmaceutical composition is administered intravenously becomes. Salt solutions and aqueous Dextrose and glycerol solutions can also as liquid carrier used, in particular for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, Sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, Glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, Propylene glycol, water, ethanol and the like. If desired the composition also small amounts of wetting agents or Emulsifiers, or pH buffering agents such as acetates, citrates or Contain phosphates. Antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, Chelating agents such as ethylenediaminetetraacetic acid and adjusting agents the tonicity such as sodium chloride or dextrose are also contemplated. The parenteral composition may be in ampules, disposable or multiple dose containers be enclosed in glass or plastic.

These Compositions can the form of solutions, Suspensions, emulsions, tablets, pills, capsules, powders, formulations with sustained release and the like. The composition can as a suppository be formulated with conventional Binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. An oral formulation may be standard carriers like about mannitol, lactose, starch, Magnesium stearate, sodium saccharin, cellulose, magnesium carbonate etc. in pharmaceutical quality contain. Examples of suitable pharmaceutical carriers are in "Remington's Pharmeceutical Sciences "by E. W. Martin described. Such compositions contain a therapeutically effective amount of the therapeutic, preferably in purified Shape, along with a suitable amount of carrier, so as to fit the shape To provide administration to the patient. The formulation should for the administration mode be suitable.

In a preferred embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizing agent and a local anesthetic such as lignocaine to relieve pain at the injection site. In general, the ingredients are provided either in unit dosage form, either separately or mixed together, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampule or pack, indicating the amount of the active ingredient. If the composition is to be infused, it may be dispensed with an infusion bottle containing sterile water or pharmaceutical grade sterile saline. If the composition is to be administered by injection, a vial of sterile water for injection or with saline so that the ingredients can be mixed before administration.

The Amount of therapeutic agent of the invention, which in the treatment of a particular disease or a particular condition is effective depends on the nature of the disease or the condition and can by standard clinical techniques be determined. additionally can optionally in vitro assays, such as those discussed in Section 5.2 can be used to help ensure optimal dosage ranges to identify. The exact one to use in the formulation Dose hangs also the route of administration and the severity of the disease or the State, and should be in accordance with the assessment of attending physician and the circumstances each patient. Suitable dosage ranges for one intravenous However, administration is generally about 20-500 micrograms Active ingredient per kilogram of body weight. Suitable dosage ranges for intranasal administration is generally about 0.01 pg / kg body weight to 1 mg / kg body weight. Effective dosages can from dose-response curves derived from in vitro or animal model test bioassays or systems, extrapolated.

The The invention also provides a pharmaceutical pack or a pharmaceutical Kit ready, comprising one or more containers filled with one or more Ingredients of the pharmaceutical compositions of the invention. Optionally, with one or more such containers, a leaflet be associated with the government agency, which is producing, Use or sale of pharmaceutical or biological Products regulated, prescribed form, with the package leaflet the approval of the authority for the Manufacture, use or sale for administration to the People reproduces.

The subsequent non-limiting Examples show the discovery of inhibition by an angiogenic Stimulus induced proliferation of endothelial cells by a Troponin subunit, and means for determining the effective dosage a troponin subunit, fragment or analogue to the Inhibition of angiogenesis, as well as for the identification of troponin subunit fragments and analogs (i.e., those fragments or analogs of a troponin subunit, which can inhibit angiogenesis). The in the examples used troponin subunit is purified as follows described.

6. EXAMPLES

Example 1: Cleaning of troponin subunit components

insulation of cardiac troponin from tissue

The procedures of Ebashi et al., 1968, J. Biochem 64: 465-477; Yasui et al., 1968, J. Biol. Chem. 243: 735-742; Hartshorne et al., 1969, Biochim. Biophys. Acta 175: 30; Schaub et al., 1969, Biochem. J. 115: 993-1004; Greaser et al., 1971, J. Biol. Chem. 246: 4226-4233; and Greaser et al., 1973, J. Biol. Chem. 248: 2125-2133, for the purification of troponin can be used. Rabbit back and leg muscles are harvested, cleaned of fat and connective tissue, and ground. The ground muscle (1 kg) is dissolved in 2 liters of a solution containing 20 mM KCl, 1 mM KHCO ₃ , 0.1 mM CaCl ₂ and 0.1 mM DTT for 5 min touched. The suspension is filtered through cheesecloth, and the washing of the residue is repeated four times. Thereafter, to the washed residue, two liters of 95% ethanol are added and the solution filtered after 10 minutes. The ethanol extraction is repeated twice. The residue is then washed three times for 10 minutes with 2 liters of diethyl ether. Finally, the residue is allowed to dry at room temperature for 2 to 3 hours.

The dried powder (from 1 kg of muscle) is extracted overnight at 22 ° C with 2 liters of a solution containing 1 M KCl, 25 mM Tris (pH 8.0), 0.1 mM CaCl ₂ and 1 mM DTT. After filtration through gauze, the residue is again extracted with 1 liter of 1 M KCl.

The extracts are combined and cooled to 4 ° C. Solid ammonium sulfate is added to produce about 40% saturation (230 g per liter). After 30 minutes, the solution is centrifuged and then 125 g of ammonium sulfate are added per liter of supernatant (60% saturation). After centrifugation, the precipitate is dissolved in 500 ml of a solution containing 5 mM Tris (pH 7.5), 0.1 mM CaCl ₂ and 0.1 mM DTT, and counterstained for 15 minutes with 15 liters of the same solution and overnight dialyzed fresh solution.

Solid KCl is added to a final concentration of 1 M and 1 M KCl solution is added to bring the volume to 1 liter. The pH is then adjusted to 4.6 by the addition of HCl, and the tropomyosin precipitate is removed by centrifugation. The pH of the supernatant is adjusted to 7.0 by KOH and 450 g ammonium sulfate added per liter (70% saturation). The precipitate is dissolved in a solution containing 5 mM Tris (pH 7.5), 0.1 mM CaCl ₂ and 0.1 mM DTT and dialyzed overnight against the same solution. Solid KCl is added to bring its concentration to 1 M, the pH is adjusted to 4.6, and the precipitate formed is removed by centrifugation. The neutralized supernatant is dialyzed against 2 mM Tris (pH 7.5) until the Nessler reaction is negative. The final yield of troponin is usually 2.5 to 3.0 g per kg of fresh muscle.

insulation of cardiac troponin from tissue

Cattle hearts are obtained about 30 minutes after death and immediately cut open, blood is rinsed off and they are dipped in ice. The left ventricle is removed, freed of excess fat and connective tissue and ground. Unless otherwise stated, all subsequent extraction and preparation steps were carried out at 0-3 ° C. The ground muscle (500 g) is homogenized for 1 min in a Waring blender, in 2.5 liters of a solution containing 0.09 M KH ₂ PO ₄ , 0.06 MK ₂ HPO ₄ , 0.3 M KCl, 5 mM 2-mercaptoethanol, pH 6.8. The homogenized muscle suspension is then stirred for 30 min and centrifuged at 1000 G for 20 min. The precipitate is re-extracted for 30 minutes and centrifuged. The residue is then washed with 2.5 liters of 5 mM 2-mercaptoethanol and centrifuged for 10 min at 1000 G, followed by two washing and centrifugation steps in succession with 1.5 liters of 50 mM KCl, 5 mM Tris-HCl (pH 8.1 ), 5 mM 2-mercaptoethanol. The residue is then washed and centrifuged twice, with 1.5 liters of 50 mM Tris-HCl (pH 8.1) and 5 mM 2-mercaptoethanol. The volume of the residue is measured and the residue is mixed with 0.5 volumes of 3 M KCl, 50 mM Tris-HCl (pH 8.1) and 5 mM 2-mercaptoethanol. After extraction for 16 to 20 hours at 0 °, the suspension is centrifuged at 15,000 G for 10 minutes. The sediment is discarded and the supernatant is adjusted to pH 7.6 with 0.05 N HCl. The filamentous precipitate that forms upon pH adjustment is removed by filtration of the extract through nylon gauze. The protein, which precipitates between 30 and 50% ammonium sulfate saturation, is collected, dissolved in a solution containing 1 M KCl and 1 mM potassium phosphate (pH 6.8) and 5 mM 2-mercaptoethanol, and added to the same solution and over 4 hours Dialyzed overnight against a fresh solution. The protein solution is clarified by centrifugation at 105,000 G for 30 minutes. The troponin is then purified by chromatography on a hydroxylapatite column, with the protein eluting between 0.08 and 0.10 M phosphate. Greaser et al., 1972, Cold Spring Harbor Symp. Quant. Biol. 37: 235-244. Rabbit heart troponin is prepared in a similar manner using a pooled batch of hearts which had been stored at -20 ° C prior to extraction.

The Troponin subunits are detected by DEAE Sephadex chromatography in 6 M urea separated. Beef Herztropomyosin gets out of the supernatant from the troponin extraction scheme with 50% ammonium sulfate saturation (see above). Ammonium sulfate will saturate to 65% added, and the precipitate is dissolved in 1 M KCl, 1 mM potassium phosphate (pH 7.0) and 5 mM 2-mercaptoethanol disbanded and dialyzed against. The protein is then purified by hydroxylapatite chromatography cleaned.

Protein determination. Protein concentrations are determined using the biuret method of Gornall et al. using bovine serum albumin as a standard certainly. Gornall et al., 1949, J. Biol. Chem. 177: 751-766.

separation of components. A sequence of SP Sephadex and DEAE Sephadex chromatography yields a complete Separation of the three cardiac troponin components.

insulation of recombinant troponin and reconstitution protocols

Troponin I and T.

DNA, the for encoding various troponin subunits and isoforms known in the art. See, for example, Wu et al., 1994, DNA Cell. Biol. 13: 217-233; Schreier et al., 1990, J. Biol. Chem. 265: 21247-21253; and Gahlmann et al. 1990, J. Biol. Chem. 265: 12520-12528.

Around to express a troponin subunit becomes one for the subunit encoding DNA into a high copy number expression plasmid such as about KP3998, using art known in the art recombinant techniques.

To express the cloned DNA, E. coli transformed with the insert-containing vector pKP1500 is grown overnight at 37 ° C, then inoculated into 4 liters of Luria-Bertani broth (LB) and incubated at 42 ° C until grown medium logarithmic phase. Isopropyl 1-thio-β-D-galactopyranoside is then added to 0.5mM and the culture is allowed to grow overnight at 42 ° C. Purification of the expressed troponin subunit, fragment or analog can be adapted from known procedures (Reinach et al., 1988, J. Biol. Chem. 250: 4628-4633 and Xu et al., 1988, J. Biol. Chem. 263: 13962-13969). The cells are harvested by centrifugation and suspended in 20 ml of 20 mM Tris, 20% sucrose, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1 mg / ml lysozyme, pH 7.5. After incubation on ice for 30 min, 80 ml of 20 mM Tris, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT are added and the cells are disrupted in a French Press (SLM Instruments). The cell residue is pelleted, the supernatant is brought to 35% in saturated (NH ₄ ) ₂ SO ₄ and stirred on ice for 30 min. After sedimentation, the supernatant is brought to 50 mM NaCl, 5 mM CaCl ₂ , 1 mM MgCl ₂ and 1 mM DTT and then to a 1.5 x 25 cm phenyl Sepharose column (Pharmacia LKB Biotechnology Inc.) loaded. The column is first washed with 50 mM Tris, 50 mM NaCl, 5 mM CaCl _2, 1 mM MgCl _2, 1 mM DTT, pH 7.5, then with 50 mM Tris, 1 mM NaCl, 0.1 mM CaCl ₂ , 1 mM DTT, pH, 7.5 until no more protein elutes. The crude troponin subunit is then eluted with 50 mM Tris, 1 mM EDTA, 1 mM DTT, pH 7.5. Fractions containing troponin subunit are pooled, dialyzed against 25mM Tris, 6M urea (United States Biochemical Corp.), 1mM MgCl ₂ , 1mM DTT, pH 8.0, and 1.5x25 cm DE52 column (Whatman) loaded. The column is eluted with a linear gradient of 0-0.6 M NaCl. From the column eluted troponin subunit, eluted fragment or analog is dialyzed against 0.1 mM NH ₄ HCO ₃ , 1 mM β-mercaptoethanol, lyophilized and stored. Purity is assessed by SDS-polyacrylamide gel electrophoresis and UV spectrophotometry. Typical yields of 6 mg of purified recombinant troponin subunit per liter of bacterial culture are expected.

The lyophilized recombinant protein is placed in a receiving buffer, consisting of 6 M urea, 20 mM Hepes (pH 7.5), 0.5 M NaCl, 2 resuspended in mM EDTA and 5 mM DTT. The mixture is allowed to stand for 1 hour Room temperature swiveled. The solution After that it lasts six hours at 4 ° C with a single exchange against a dialysis buffer consisting of 0.5 M NaCl, 20 mM Hepes (pH 7.5) and 0.5 mM DTT dialyzed.

The Protein concentration is for each subunit determined at 280λ. The extension coefficient of troponin I is 0.40, and that of troponin T is 0.50.

Troponin C

The lyophilized recombinant protein is placed in a receiving buffer, consisting of 0.1 M NaCl, 20 mM Hepes (pH 7.5), 2 mM EDTA and 5 resuspended in mM DTT. This solution will last for six hours at 4 ° C with a single exchange against a dialysis buffer of 0.1 M NaCl, 20 mM Hepes (pH 7.5) and 0.5 mM DTT dialyzed.

The Protein concentration is determined by measuring absorbance at 280λ. The extension coefficient for Troponin C is 0.18.

reconstitution of combined subunits

Protein concentrations with the same molar reconstitution ratios of troponin subunits C, I and T are obtained for all different combinations. These concentrations of the respective proteins are combined in a reconstitution buffer consisting of 0.1 M NaCl, 0.1 M CaCl ₂ , 5 mM DTT, 5 mM Hepes (pH 7.5). Dialysis is carried out for 20-24 hours at 4 ° C, against a dialysis buffer consisting of 0.1 M NaCl, 0.1 M CaCl ₂ , 0.5 mM DTT and 5 mM Hepes (pH 7.5), with three times buffer change.

The Protein concentration approximates by measuring absorbance at 278λ certainly. The troponin trimer has an extension coefficient from 0.45 at 278λ.

Example 2: Inhibition the proliferation of endothelial cells as measured by DNA synthesis

The Inhibitory effect of a troponin subunit, a fragment or analogous to the proliferation of bFGF-stimulated EC according to the following Procedure to be determined.

DNA synthesis of endothelial cells

On day one, 5,000 capillary cattle endothelial cells in DMEM / 10% CS / 1% GPS are plated in each well of a previously gelatinized 96-well tissue culture plate. On day two, the cell medium is changed to DMEM, 2% CS, 1% GPS, 0.5% BSA (complete medium) supplemented with 10 μl of 1 mg / ml "cold" thymidine per 50 ml of medium. On day three, test samples are added in complete media in two batches. Additionally, except for the appropriate controls, bFGF is added to each well at a final concentration of 0.2 ng / well. On day four, 5 μl of 1:13 diluted ³ H-thymidine stock solution is added to each well and the plate is incubated for 5-6 hours. After incubation, the medium is aspirated and the residue is rinsed once with PBS, then twice for 5 minutes with methanol, followed by two rinses for 10 minutes each with 5% TCA. The cell contents of the wells are then rinsed three times with water, dried on the plate, and 100 μl of 0.3 N NaOH is added to each well. The contents of the well are then transferred to scintillation vials and 3 ml of Ecolume added to each vial. The samples are then counted on the scintillation counter.

DNA synthesis of 3T3 cells

The DNA synthesis in bFGF-stimulated 3T3 cells provides a control with the the for proliferation of bFGF-stimulated Endothelial cells obtained results can be evaluated. The DNA synthesis in the 3T3 cells can be done according to the following method be determined.

BALB / c 3T3 cells are trypsinized and resuspended at a concentration of 5 x 10 ⁴ cells / ml. 200 μl aliquots are plated in 0.3 cm ² microtiter wells (Microtest II tissue culture plates, Falcon). After reaching confluency, in a period of 2 to 3 days, the cells are incubated for at least another 5 days to deplete the medium of growth promoting factors. These growth conditions give confluent monolayers of non-dividing BALB / c 3T3 cells. Test samples are dissolved in 50 μl of 0.15 M NaCl and added to the microtiter wells together with [ ³ H] TdR. After incubation for at least 24 hours, the medium is removed and the cells are washed in PBS. Cell fixation and removal of unincorporated [ ³ H] TdR is effected by the following sequential steps, adding methanol twice for periods of 5 minutes, 4 washes with H ₂ O, adding cold 5% TCA twice for periods of 10 And 4 washes with H ₂ O. DNA synthesis is measured by either counting liquid scintillation or autoradiography using a modification of the method described by Haudenschild et al., 1976, M. Exp. Cell Res. 98: 175 , For scintillation counting, cells are lysed in 150 μl of 0.3 N NaOH and counted in 5 ml Insta-Gel liquid scintillation cocktail (Packard) using a Packard Tri-Carb liquid scintillation counter. Alternatively, autoradiography can be used to quantify DNA synthesis by breaking the bases of the microtiter wells and attaching them to glass slides with sialastic glue. The slides are dipped in a 1 g / ml solution of NTB2 Nuklear Track Emulsion (Kodak) and exposed for 3-4 days. The emulsion is developed for 10 minutes with Microdol-X solution (Kodak), rinsed with distilled H ₂ O, and fixed for three minutes with Rapid Fixer (Kodak). The autoradiograms are stained with a modified Giemsa stain. At least 1000 nuclei are counted in each well and DNA synthesis expressed as the percentage of labeled nuclei. Cell division is measured by counting the number of cells in microtiter wells, using a grid, after 40-48 hours of incubation with test samples.

Example 3a Inhibition the proliferation of endothelial cells, measured by colorimetric Determination of activity of cellular acid phosphatase and electronic cell count

One rapid and sensitive screening for the inhibition of EC proliferation in response to treatment with a troponin subunit, an analog or derivative of the invention comprises incubating of cells in the presence of varying concentrations of Inhibitors and the determination of the number of endothelial cells in culture based on the colorimetric determination of the activity of cellular acid Phosphatase described by Connolly et al., 1986, J. Anal. Biochem. 152: 136-140.

We reasonably the effect of troponin on the proliferation of capillary endothelial cells (EC) in an assay that has the ability of this protein, stimulating the proliferation of endothelial cells impaired by a known angiogenesis factor (bFGF), measures.

On day 1, capillary endothelial cells and Balb / c 3T3 cells are plated separately (2 x 10 ³ / 0.2 ml) on 96-well gelatin-coated tissue culture dishes. On day 2, the cells were resumed Nutrients, with Dulbecco's modified Eagle's medium (Gibco) with 5% calf serum (Hyclone) (DMEM / 5) and bFGF (10 ng / ml) (FGF Co.) and increasing concentrations of the troponin subunit. These substances were added simultaneously, in volumes not exceeding 10% of the final volume. Wells containing phosphate buffered saline (PBS) (Gibco) alone and PBS + bFGF were included as controls. On day 5, the medium was removed and the cells were washed with PBS and dissolved in 100 μl buffer containing 0.1 M sodium acetate (pH 5.5), 0.1% Triton X-100 ^™ and 100 mM p-nitrophenyl phosphate ( Sigma 104 phosphatase substrate), lysed. After two hours of incubation at 37 ° C, the reaction was stopped by adding 10 μl of 1 N NaOH. Color development was determined using a fast microplate reader (Bio-Tek) at 405 nm.

The percent inhibition was determined by comparing the numbers cells from wells exposed to the stimulus with those exposed to stimulus and troponin subunits.

It All three troponin subunits were found to proliferate bFGF-stimulated EC inhibited as by colorimetric Assays measured.

Troponin C inhibited the proliferation of bFGF-stimulated endothelial cells in a dose-dependent manner at all concentrations tested ( 1 ). The percent inhibition of bovine endothelial cell proliferation ("BCE") was 54%, 86%, 83% and 100%, respectively, at concentrations of 280 nM, 1.4 μM, 2.8 μM and 5.6 μM, respectively. An inhibition of 100% was observed at a concentration of 20 μg / well (5.6 μM). IC ₅₀ represents the concentration at which 50% inhibition of growth factor bFGF-induced stimulation was observed. It was determined that the IC ₅₀ of troponin C is 278 nM.

Troponin I inhibited bFGF-stimulated BCE proliferation in concentrations of 1 and 5 μg / well, but no inhibition was observed in the sample tested at 10 μg / well ( 2 ). The percent inhibition of BCE was 33% and 46%, respectively, at concentrations of 240 nM and 1.2 μM, respectively. It was determined that the IC ₅₀ of troponin I is 1.14 μM.

Troponin T inhibited bFGF-stimulated EC proliferation at concentrations of 10 and 20 μg / well, but not at concentrations of 1 and 5 μg / well ( 3 ). The proliferation of BCE was inhibited at 1.6 μM and 3.3 μM, respectively, at 23% and 62%, respectively. The IC ₅₀ of troponin T was determined to be 2.14 μM.

The combination of troponin subunits C and I inhibited EC at all concentrations tested ( 4 ). The percent inhibition of BCE proliferation was 52%, 54%, 73% and 47% at 130 nM, 645 nM, 1.3 μM and 2.6 μM, respectively. It was determined that the IC _{50 of} this combination is 110 nM.

It has been observed that the combination of troponin subunits C, I and T at a concentration of 360 nM (5 μg / well, 5 ) inhibited bFGF-stimulated BCE proliferation by 16%.

The tested troponin samples had no detectable inhibitory Effect on the growth of Balb / c 3T3 cells, a non-endothelial Cell type.

Example 4: Inhibition the migration of capillary endothelial cells by troponin

A Determination of the ability the troponin subunit, the derivative or analog, the angiogenic Process of capillary EC migration in response to an angiogenic Can inhibit stimulus, using a modification Boyden chamber technique to determine the effect of the troponin subunit, of the derivative or analog for capillary EC migration. Falk et al., 1980, J. Immunol. 118: 239-247 (1980). A blind well boyden chamber consists of two (one upper and one lower) depression, the through a porous Membrane are separated. J. Exp. Med. 115: 453-456 (1962). An acquaintance Concentration of growth factor is in the lower wells given and a predetermined number of cells, and troponin subunit, Derivative or analog is added to the upper wells. cells, those on the upper surface cling to the membrane, migrate through the membrane and adhere on its lower surface at. The membrane can then be fixed and stained for counting, using the method of Glaser et al., 1980, Nature 288: 483-484.

Migration is measured using blank wells (Neuroprobe, No. 025-187) and polycarbonate membranes with pores of 8 microns (nucleopore) pre-coated with fibronectin (6.67 μg / ml in PBS) (human, Cooper). Basic FGF (Takeda Co.) diluted in DMEM with 1% Käl Berserum (DMEM / 1) is added at a concentration of 10 ng / ml to the lower well. The upper wells receive 5 x 10 ⁵ capillary EC / ml, and increasing concentrations of purified troponin subunit, derivative or analog, used within 24 hours of purification. Control wells receive DMEM / 1, either with or without bFGF. The migration chambers are incubated for 4 hours at 37 ° C in 10% CO ₂ . The cells on the upper surface of the membrane are then wiped off by pulling the membrane over a wiper blade (Neuroprobe). The cells which have migrated through the membrane to the lower surface are fixed in 2% glutaraldehyde followed by methanol (4 ° C) and stained with hematoxylin. Migration is quantified by counting the number of cells on the bottom surface in 16 oil immersion fields and comparing this number with that obtained for the control.

Example 5: Inhibition tumor growth as determined by a SCID mouse model system

The effects of recombinant troponin I on the growth of PC-3 prostate carcinoma cells were determined in immunodeficient (SCID) mice in a treatment group and a control group of four mice each. A dorsal subcutaneous implant of 10 ⁶ PC-3 cells was made and observed until a volume between 100-400 mm ^{3 was} reached. After the tumor had reached the threshold volume, subcutaneous injections of 50 mg / kg recombinant troponin I were started twice a day in the treatment group.

6 shows that 28 treatment days resulted in a reduction of approximately 50% of the tumor volume in the treatment group compared to the tumor volume of the control group.

Example 6: Inhibition in vivo neovascularization by troponin as determined by the mouse corneal bag assay

A pellet of sucrose octasulfate, Hydron ^™ and basic fibriblast growth factor (40 ng / pellet) was placed in a mouse corneal microtube. The mouse received subcutaneous injections of recombinant troponin I, 50 mg / kg every 12 hours, starting 48 hours before implantation.

Corneal angiogenesis was evaluated by slit lamp microscopy. On day 6, angiogenesis leads into Control eyes to vessels that extend into the pellet on day 6. In the treated animals At this time, a 50% reduction in the density of Blood vessels and a 30% inhibition of length observed by blood vessels.

SEQUENCE LISTING

Claims (14)

A pharmaceutical composition comprising an amount of at least one peptide capable of effectively inhibiting angiogenesis, and a pharmaceutically acceptable carrier therefor, characterized in that the peptide is a troponin subunit selected from the group consisting of subunits C, I and T, is. Pharmaceutical composition according to claim 1, characterized in that the composition is additional an effective amount of one different from the troponone subunit molecule, which negatively regulates angiogenesis. Pharmaceutical composition according to claim 1 or 2, characterized in that the peptide a. is an inhibitor of bFGF-stimulated bovine endothelial cell proliferation with an IC ₅₀ of less than or equal to 10 μM; b. has a length of more than 75 amino acids; and c. a homology greater than 80% to a subunit selected from the group consisting of human fast twitch troponin subunit C (SEQ ID NO: 1), human fast twitch troponin subunit I (SEQ ID NO: 1); NO: 2), and human fast-twitch troponin subunit T (SEQ ID NO: 3). Composition according to Claim 3, characterized that the peptide is a mammalian troponin subunit, selected from the group consisting of human, bovine, troponin subunits Rabbit, mouse and rat. Composition according to Claim 3 or 4, characterized that the peptide has a homology of more than 80% to the human fast-twitch Troponin subunit C or the human fast-twitch troponin subunit I has. Composition according to Claim 3, characterized that the peptide has a homology of more than 95% to a human Troponin subunit has. Composition according to Claim 6, characterized the human troponin subunit is the human fast-twitch troponin subunit C or the human fast-twitch troponin subunit I. A composition according to any one of claims 1 to 7, characterized in that the carrier for topical application the eye or the skin is acceptable. A composition according to any one of claims 3 to 7, characterized in that the angiogenesis inhibitor in a biodegradable, biocompatible, polymeric delivery device is. Peptide for use as a drug for inhibition angiogenesis, characterized in that the peptide is a troponin subunit, selected from the group consisting of the subunits C, I and T, is. Peptide according to claim 10, characterized in that the peptide a. is an inhibitor of bFGF-stimulated bovine endothelial cell proliferation with an IC ₅₀ of less than or equal to 10 μM; b. has a length of more than 75 amino acids; and c. a homology greater than 80% to a subunit selected from the group consisting of human fast twitch troponin subunit C (SEQ ID NO: 1), human fast twitch troponin subunit I (SEQ ID NO: 1); NO: 2), and human fast-twitch troponin subunit T (SEQ ID NO: 3). Use of a peptide for the manufacture of a medicament for the Inhibition of angiogenesis in a living organism, the one with angiogenesis associated disease or disorder has, being the disease or disorder is a solid tumor or is a tumor of the central nervous system, or an ophthalmic Illness or disorder, or hemangioma, Arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, Granulations, hemophilic Joints, hypertrophic scars, poor fracture healing, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions, and wherein the subject is administered an effective amount of a peptide characterized in that the peptide is a troponin subunit, selected from the group consisting of the subunits C, I and T, is. Use according to claim 12, characterized in that the peptide a. is an inhibitor of bFGF-stimulated bovine endothelial cell proliferation with an IC ₅₀ of less than or equal to 10 μM; b. has a length of more than 75 amino acids; and c. a homology greater than 80% to a subunit selected from the group consisting of human fast twitch troponin subunit C (SEQ ID NO: 1), human fast twitch troponin subunit I (SEQ ID NO: 1); NO: 2), and human fast-twitch troponin subunit T (SEQ ID NO: 3). Use according to claim 13, characterized that the peptide has a homology of more than 80% to the human fast-twitch Troponin subunit C or the human fast-twitch troponin subunit I has.